Although it is well known that women are more susceptible to the toxic effects of ethanol (EtOH) than men, much less is known about the molecular mechanisms underlying alcohol toxicity in women especially as relates to bone. Alcohol abuse during early adulthood results in impaired bone growth and in the U.S.A. approximately 20% of women age 18-30 (4.4 million) binge drink. A resulting reduction in peak bone mass would predispose women to osteoporosis in later life. The molecular mechanisms underlying the toxic effects of EtOH on bone remain poorly understood. EtOH, particularly at higher concentrations characteristic of binge drinking, can increase bone resorption. We have developed a new mouse model to study alcohol-induced bone loss in cycling females. Chronic consumption of EtOH as part of liquid diets using this system produces EtOH-induced bone loss without compromising food intake. Bone loss in female mice was accompanied by increased serum markers of bone resorption and increased osteoclast numbers in ex-vivo bone marrow cultures. These data are consistent with previously published data from our laboratory in cycling female rats fed ethanol via total enteral nutrition. In the rat model, increased bone resorption was accompanied by increased expression of RANKL in bone. RANKL is a member of the TNF family expressed on the surface of osteoblasts which signals through the receptor RANK on the surface of osteoclast precursors to stimulate osteoclastogenesis. EtOH-induction of RANKL occurred in primary osteoblast cultures from rats and mice and in rat and mouse UMR-106 and ST-2 osteoblast-like cell lines. EtOH-induction of RANKL was blocked by the antioxidant N-acetylcysteine (NAC) in vitro. RANKL appears to be regulated via ERK1/2 and STAT3 phosphorylation. In vitro data suggest that the ERK/STAT3/RANKL pathway is stimulated by reactive oxygen species (ROS) produced by an ethanol-induced NADPH oxidase (NOX4). Expression of this enzyme in osteoblasts is increased by EtOH and blocked by NAC. The mouse liquid diet model will be utilized to determine the role of NOX4 and oxidative stress in EtOH-induced bone resorption in vivo using p47phox -/- mice and catalase transgenic mice. In vitro, the mouse ST-2 cell line, differentiated mouse stromal osteoblast cultures and osteoblast/osteoclast precursor co-cultures will be utilized to probe the molecular cascade from EtOH to RANKL and the inhibition of EtOH-induced RANKL by NAC and other antioxidants. NOX gain and loss of function studies will be performed by transient transfection and use of siRNA and the effects of hydrogen peroxide and free radical scavenging on the ERK/STAT3/RANKL cascade will be examined. In addition, in vivo studies will be conducted to examine the ability of antioxidants to block EtOH-induced bone resorption.